spectrophotometric method for the determination of drugs

ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry

http://www.e-journals.net 2010, 7(2), 395-402

Spectrophotometric Method for the Determination of

Drugs Containing Phenol Group by Using

2, 4- Dinitrophenylhydrazine

PADMARAJAIAH NAGARAJA* and ASHWINEE KUMAR SHRESTHA

Department of Studies in Chemistry, University of Mysore,

Manasagangotri, Mysore-57006, Karnataka, India.

[email protected]

Received 2 August 2009; Accepted 1 October 2009

Abstract: A spectrophotometric method has been proposed for the

determination of four phenolic drugs; salbutamol, ritodrine, amoxicillin and

isoxsuprine. The method is based on the oxidation of 2, 4- dinitrophenyl-

hydrazine and coupling of the oxidized product with drugs to give intensely

colored chromogen. Under the proposed optimum condition, beer’s law was

obeyed in the concentration range of 2.5-17, 2-29, 4-33 and 5-30 µg/mL for

salbutamol, ritodrine, amoxicillin and isoxsuprine respectively. The limit of

detection (LOD) and limit of quantification (LOQ) were 0.2, 0.83, 0.09,

0.84 µg/mL and 0.66, 2.79, 0.3 and 2.81 µg/mL in the same order. No

interference was observed from common pharmaceutical adjuvants. The

ringbom plots and low relative standard deviation assert the applicability of

this method. The suggested method was further applied for the determinations

of drugs in commercial pharmaceutical dosage forms, which was compared

statistically with reference methods by means of t- test and F- test and were

found not to differ significantly at 95% confidence level. The procedure is

characterized by its simplicity with accuracy and precision.

Keywords: 2, 4- Dinitrophenylhydrazine, Salbutamol sulfate, Ritodrine HCl, Amoxicillin trihydrate,

Isoxsuprine HCl, Spectrophotometry

Introduction

Salbutamol sulfate (SLB), ritodrine HCl (RTD), amoxicillin trihydrate (AMOX) and

isoxsuprine HCl (IXP) are frequently prescribed biologically active compounds. Salbutamol

sulfate and ritodrine is beta-2 agonist agent. SLB is used as a bronchodilator in the

management of reversible airway obstruction. RTD is a uterine relaxant drug used to arrest

premature labour. Amoxicillin trihydrate (AMX) is a broad spectrum antibiotic agent acting

396 P. NAGARAJA et al.

on both gram positive and gram negative bacteria. Isoxsuprin, a derivative of adrenaline, is

an alpha receptor antagonist and beta receptor agonist, which is used as vasodilator and

uterine relaxant.

Among the various methods available for the estimation of these drugs such as HPLC,

Electrophoresis, Gas chromatography, Spectrophotometry method is still a preferred

technique due to its simplicity and economy. Several colorimetric methods are available for

the estimation of salbutamol1-6

, ritodrine6-12

, amoxicillin13-16

and isoxsuprine6, 17-18

.

The present work is an attempt to develop a rapid and sensitive method for the colorimetric

estimation by 2, 4 dinitrophenylhydrazine (2, 4 DNP). 2, 4 DNP, also known as Brady’s

reagent, has been used for the characterization of aldehyde and ketones by hydrazone formation.

We report here its application in the estimation of phenolic drugs. The method is based on the

oxidation of 2, 4 DNP to produce diazonium cation, which couples with phenolic drugs to yield

highly absorbing chromagen. Studies on different variables affecting the reaction were

optimized. The developed method is simple, accurate and applicable for their determination in

pharmaceutical formulations. All the above mentioned drugs are official in BP 2005.

Experimental

A JASCO (Model UVIDEC-610 UV-VIS spectrophotometry with 1cm matched quartz

cuvettes was used for all absorbance measurements.

Reagents and solutions

Analytical reagent grade chemicals and double distilled water were used throughout the

experiment. Salbutamol sulfate (Cipla, India), ritodrine HCl (Duphar-Interfran Ltd, India),

amoxicillin trihydrate (Sigma Aldrich, USA) and isoxsuprine HCl (Sigma Aldrich, USA)

were procured and used as received. Stock solutions of each drug containing 100 µg/mL

were prepared by dissolving 10 mg of the respective drugs in 100 mL of water. The

solutions were further diluted quantitatively according to their linearity range. The

pharmaceutical preparations were purchased from a local market and analysed.

Preparation of 2, 4-DNP reagents

A 0.04% w/v and a 0.08% w/v of the reagent solution were freshly prepared by dissolving 0.04 g

and 0.08 g of 2, 4 DNP (SRL Chemicals, India) in 1 mL and 2 mL of concentrated H2SO4

respectively and diluting to 100 mL with water.

Preparation of oxidizing agents

For amoxicillin, a 0.15% w/v potassium periodate (The British Drug House, England) and

for other drugs, a 4% w/v potassium iodate (Loba-Chemie, India) solution were prepared

by dissolving suitable quantities in water.

Preparation of tablet/capsule sample solution

Twenty tablets of each drug was weighed, powdered and mixed thoroughly. Similarly, ten

capsule of AMX were carefully evacuated, and mixed. A quantity equivalent to 10 mg of each

drug was transferred to 100 mL volumetric flask. The drugs were dissolved in water, shaken

well, sonicated and made up to the volume with water. The resultant solutions were filtered and

analysed as described under general procedure.

General procedure

Accurately measured suitable volume of SLB, RTD and IXP were transferred from stock

solution to 10 mL volumetric flasks, which could be diluted quantitatively to obtain 2.5-17,

Spectrophotometric Method for the Determination of Drugs 397

2-29 and 5-30 µg/mL respectively. To each flask containing drugs in the order mentioned

above, 1, 2 and 1.5 mL of 2, 4 DNP (0.04%) and 2, 4 and 1.5 mL of KIO3 (4%) were

added, which were made alkaline by adding 1 mL each of NaOH (10 N). Similarly, for

AMOX, an accurate quantity of drugs that could be diluted to 4-33 µg/mL was transferred in

10 mL volumetric flasks followed by addition of 1.5 mL of 2, 4 DNP (0.08%), 1.5 mL of

KIO4 (0.15%) and 0.5 mL of NaOH (10 N). The red color hence developed was further

diluted to the volume with water.

Results and Discussion

Spectral characteristic

The absorption spectra of the reaction product of oxidized 2, 4 DNP with drugs show

maximum absorption (λmax) at 540, 510, 520 and 520 nm for SLB, RTD, AMOX and IXP

respectively. The blank solution was slightly yellowish in color that had negligible

absorbance at the λmax in which the drugs were analysed. The thus formed color was stable

for more than two hours. A temperature range of 20-30 0C is preferred for the reaction.

Reaction sequence and stoichiometric relationship

The 2, 4 DNP is oxidized by KIO3 or KIO4 to give diazonium cation that reacts with drugs

by electrophilic substitution at the phenolic ring to give deep colored chromogens. The

proposed reaction sequence for RTD is shown in Figure 1. The reaction sequence for other

drugs is expected to follow the same sequence. The combining ratio was evaluated by the

Job’s method of continuous variation and molar ratio method, which shows that the complex

formed between the drugs, SLB, RTD, IXP and AMX with 2, 4 DNP were 1:1, 1:2, 1:1 and

1:1 respectively. The plot of Log [Absorbance] against Log [2, 4 DNP] and the drugs in

limiting logarithmic method further confirmed the result. Figure 2 shows the continuous

variation plot and limiting logarithmic plot for RTD.

Figure 1. Proposed reaction mechanism.

b


Figure 2.(a) Job’s method of continuous variation plot and (b) Limiting logarthimic plot

for the molar reactivity for RTD. The concentration of RTD and 2, 4 DNP is 2×10-3

M.

Optimum reaction condition

By varying one and keeping other experimental parameters and the amount of drug constant, the

effect of 2, 4-DNP, oxidizing agents and NaOH were studied. Maximum color intensity was

obtained when 0.8-1.3, 1.8-2.1, 1.3-1.5 mL of 2, 4 DNP and 1.8-2.1, 3.7-4.2, 1.5-1.7 mL of iodate

were added to SLB, RTD and IXP respectively. The amoxicillin produced more intense color when

Potassium periodate was used as an oxidizing agent. The optimum quantity for amoxicillin was

found to be 1.4-1.5 mL of 2, 4 DNP and 1.3-1.6 mL of potassium periodate. Different

concentrations of sodium hydroxide was used for maximum color development and was found that

1 mL of 10 N NaOH was optimum except for AMX in which 0.5 mL of 10N NaOH was needed.

During the analysis, the quantities of the reagents as mentioned in general procedure were taken.

Validation of the proposed method

Linearity, detection and quantification limit

Calibration graphs were constructed using standard solutions under optimum experimental

condition. A linear relationship was observed between the absorbance and concentration of

drugs from 2.5-17, 2-29, 4-33 and 5-30 µg/mL for SLB, RTD, AMOX, and IXP respectively.

The molar absorptivity and sandell’s sensitivity for each drug were calculated from beer’s law.

The Ringbom plots demonstrated the range of 3-16, 5-26, 5-31 and 7-25 µg/mL for SLB, RTD,

AMOX and IXP respectively. The graph showed a negligible intercept, which was calculated

by the least-square method’s regression equation:

A = a + bc

[RTD]/[RTD]+[2,4 DNP]

Ab

sorb

ance

(a)

log [2,4 DNP]

log

Ab

sorb

ance

log [RTD]

log

Ab

sorb

ance

(b)


where, A is the absorbance of solution in a 1 cm cell, a is the intercept, b is the slope, and

c is the concentration of the measured solution in µg/mL. The high molar absorptivities of the

resulting colored solution indicated high sensitivity of the method. The limit of detection

(LOD) and the limit of quantification (LOQ) value were determined using the formula

LOD or LOQ = К SD/b

where, К = 3 for LOD and 10 for LOQ, SD and b stand for standard deviation of the

blank and slope, respectively. Repeatability and level of precision was tested by analyzing

six replicate samples containing 13, 15, 20 and 16 µg/mL and was found to be adequate for

quantification of drugs as shown by their relative standard deviation. The confidence limits

for the slope of line of regression and the intercept were computed using the relation, b±tSb

and a±tSa at 95% confidence level. The results are shown in Table 1. The error (Sc) in the

determination of a given concentration of drugs was defined by the expression where,

Sc =b

Sy/x2/1

22

2

)(

)(11

−

−

++

∑ xxb

yyo

n

y and x are the average values of the absorbance and concentration, respectively, for n

standard samples. It is clear from the graph that the error is reached minimum when

absorbance corresponding to about 10, 15, 13 and 15 µg/mL of SLB, RTD, AMOX and IXP

respectively, when the actual absorbance is equal to the average absorbance. The plot of

error, Sc vs. concentration of RTD is shown in Figure 3.

Table 1. Optical characteristics and statistical data of the regression equation for the reaction

of the proposed method.

Parameters Optical Characteristic

SLB RTD AMOX IXP

Colour Reddish Reddish Reddish Reddish

λmax, nm 540 510 520 520

Beer’s Law range, µg/mL 2.5-17 2-29 4-33 5-30

Molar absorptivity, L/mol/cm ×104 2.19 0.741 1.0 8.04

Sandell’s sensitivity, µg/cm2 0.0263 0.0436 0.0418 0.0419

Limit of Detection, µg/mL 0.2 0.837 0.09 0.8451

Limit of quantification, µg/mL 0.66 2.79 0.3 2.817

Ringbom plot range, µg/mL 3-16 5-26 5-31 7-25

Regression Equation (Y)a

Slope (b) 0.036 0.0252 0.0221 0.0275

Standard deviation of Slope(Sb) ×10-4

4.8 8.35 2.66 4.198

Intercept (a) 0.0129- 0.0125 0.0151 0.0424

Standard deviation of Intercept (Sa)×10-3

5.84 14 5.0 7.88

Correlation Coefficient(r) 0.9995 0.9993 0.9987 0.9993

Relative standard deviationb 0.185 0.349 0.223 0.236

±tsb ×10-3

1.33 2.31 7.36 1.165

±tsa

0.0152 0.0387 0.0138 0.02187 aA = a + bc, where c is the concentration of the measured solution in µg/mL. ±tsb= Confidence

interval for Slope at 95% Confidence Level, ±tsa= Confidence interval for Intercept at 95%

Confidence Level b Average of six determination (concentration of 13, 15, 20 and 16 µg/mL of pure

drugs of SLB, RTD, AMX, and IXP respectively).


Figure 3. Plot of error in the determination of RTD

Interference studies

The effect of common excipients used in the pharmaceutical preparation were studied by analyzing synthetic sample solutions containing the quantity of drugs as mentioned in Table 2 in presence of 100 fold more concentration of each excipients. The tolerance limit was defined as the concentration which gave an error of ±3.0% in the determination of drugs. The common excipients such as sodium chloride, starch, dextrose, lactose, talc, carboxymethyl cellulose, magnesium stearate, sucrose, had no effect in the analysis.

Table 2. Recovery of drugs from solution in presence of a 100 fold concentration of various

additives used as excipients in formulation.

Excipients Recovery±R.S.Da

SLBb RTD

c AMOX

d IXP

e

Dextrose 99.9±0.7 99.9±0.5 99.8±0.5 99.8±0.4

Lactose 100±0.2 99.9±0.2 99.9±0.4 99.8±0.1

Sucrose 99.9±0.5 99.9±0.1 99.8±0.2 99.9±0.4

Starch 99.9±0.1 99.8±0.4 99.9±0.1 99.9±0.3

Talc 99.8±0.4 99.9±0.3 99.9±0.4 99.7±0.0

Carboxymethyl cellulose 99.8±0.4 99.8±0.5 99.8±0.5 99.8±0.3

Magnesium Sterate 99.8±0.5 99.7±0.7 99.8±0.5 99.7±0.5

Sodium Chloride 100±0.1 100±0.1 100±0.2 100±0.2 aMean of 3 determination, b Concentration of SLB used – 10 µg/mL, c Concentration of RTD used– 15 µg /mL, d Concentration of AMOX used– 15 µg /mL, e Concentration of IXP used– 15 µg /mL

Precision studies

The short term precision (intraday precision) of the drugs were evaluated by measuring 5 independent samples at 3 different concentration levels (4.0, 8.0, 12.0 µg/mL for SBM, 10.0, 15.0, 20.0 µg/mL for RTD, 15.0, 20.0, 25.0 µg/mL for AMOX, and 15.0, 20.0, 25.0 µg/mL for IXP). Similarly, the assay for daily precision (interday precision) at the same concentration level was repeated for 5 consecutive days (Table 3). The available pharmaceutical dosage forms of the investigated drugs were analysed by the proposed method. The precision of the method was checked by taking six replicate measurements. The results obtained by the proposed and the reference methods for the dosage forms were compared statistically by means of F- and t- test and were found not to differ significantly at 95% confidence level. The reliability and accuracy of the proposed method were further ascertained through recovery studies using the standard addition method by adding different amount of standard drugs to the preanalyzed dosage forms such that the cumulative amount after adding the drugs did not exceed their linearity range (Table 4).

Concentration of RTD, µg/mL

Err

or,

Sc


Table 3. Intraday and interday precision data.

Formulation Amount taken,

µg/mL

Intraday,

%Recovery ±R.S.Da

Interday,

%Recovery ±R.S.Db

SBM 4.0 3.99±0.1 3.98±0.1

8.0 8.01±0.3 7.98±0.3

12.0 12.0±0.2 12.07±0.5

RTD 10 9.99±0.5 9.97±0.7

15 14.97±0.6 15.01±0.9

20 20.07± 1.2 20.10±0.9

AMOX 15 15.0±0.7 14.99±0.9

20 19.98±0.3 20.0±0.9

25 25.0±0.5 25.03±0.5

IXP 15 15.0±0.4 15.05±0.7

20 20.01±1.2 20.09±1.2

25 25.02±0.8 25.07±1.3 a Mean of 5 determinations, b Mean of 5 determinations performed over a period of 5 days

Table 4. Analysis of drugs in pharmaceutical formulations.

Formulation Labelled,

mga

Amount found by

proposed method±S.D,

mga

Reference

method±S.D a*

% Recovery by

proposed methodsb

Asthalinc 4 3.96±0.032

F = 1.64

T = 0.94

3.98±0.041 (6) 99.0±0.3

Yutopard 10 9.97±0.12

F = 1.5

T = 0.51

10.01±0.15 (11) 99.7±0.33

Mox 250e 250 250.0±0.14

F = 1.36

T = 0.66

249.87±0.12 (14) 100.00±0.02

Duvadilanf 10 10.00±0.25

F = 1.34

T = 1.2

9.81±0.29 (6) 99.9±0.36

a Average ± standard deviation of six determination; the t- and F- values obtained after comparison to the

reference methods have following theoretical values at 95% confidence limit; t= 2.44 F= 5.05, bAfter

adding four different amounts of pure drugs to the fixed concentration of preanalysed pharmaceutical

formulations, cSBM equivalent to 4 mg/tablet (Cipla, India), dRTD equivalent to 10 mg/tablet (Alimbic,

India), eAMX equivalent to 250 mg/Capsule (Ranbaxy, India), fIXP equivalent to 10 mg/tablet Tablet

(Duphas Interfran, India)*References inside the bracket indicated reported methods given under references.

Conclusion

We have proposed a new spectrophotometric method for the determination of phenolic drugs,

which is fairly sensitive, simple, and economical with reasonable precision and accuracy.

The optical parameters and statistical comparison justify this method for application in

routine drug estimation in pure and dosage forms. Also, the procedures do not involve any

critical reaction conditions or tedious sample preparation steps. So, the recommended

method is well suited for the assay and evaluation of drugs in pharmaceutical preparation

and can also be considered as a general method for the quantification of phenolic drugs.


Acknowledgment

One of the authors Ashwinee Kumar Shrestha thanks the Jawaharlal Nehru Memorial Fund,

India (Ref: SU/2/552/2008-09/760) for providing scholarship and University of Mysore for

providing facilities for the investigation.

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